Ploidy levels in diverse picocyanobacteria from the Baltic Sea

Abstract In nature, the number of genome or chromosome copies within cells (ploidy) can vary between species and environmental conditions, potentially influencing how organisms adapt to changing environments. Although ploidy levels cannot be easily determined by standard genome sequencing, understanding ploidy is crucial for the quantitative interpretation of molecular data. Cyanobacteria are known to contain haploid, oligoploid, and polyploid species. The smallest cyanobacteria, picocyanobacteria (less than 2 μm in diameter), have a widespread distribution ranging from marine to freshwater environments, contributing significantly to global primary production. In this study, we determined the ploidy level of genetically and physiologically diverse brackish picocyanobacteria isolated from the Baltic Sea using a qPCR assay targeting the rbcL gene. The strains contained one to four genome copies per cell. The ploidy level was not linked with phylogeny based on the identity of the 16S rRNA gene. The variation of ploidy among the brackish strains was lower compared to what has been reported for freshwater strains and was more similar to what has been reported for marine strains. The potential ecological advantage of polyploidy among picocyanobacteria has yet to be described. Our study highlights the importance of considering ploidy to interpret the abundance and adaptation of brackish picocyanobacteria.


INTRODUCTION
Ploidy, the number of genome copies within one cell/ chromosome, is a well-known phenomenon in Eukarya and can be found among organisms ranking from ciliates to humans (Comai, 2005;Thorpe et al., 2007;Wendel, 2000).For a long time, prokaryotes were assumed to be haploid (to contain only one circular genome per cell).However, it is now established that oligoploidy (multiple genome copies per cell) and polyploidy (≥10 genome copies per cell) are common among the Bacteria and Archaea domains (Pecoraro et al., 2011;Soppa, 2014;Suh et al., 2001;Tobiason & Seifert, 2006).In recent years, the number of studies sampling environmental DNA and using molecular tools (omics) to characterize microbial communities has increased drastically (Farrant et al., 2016, Sunagawa et al., 2015;Tully et al., 2018).In such studies, the relative abundances of organisms are often estimated based on the assumption that there is only one genome copy per organism.Gaining information about the variation in genome copy numbers in specific organism groups and factors that influence ploidy is therefore of high importance for accurate community composition studies.
Cyanobacteria, photosynthetic prokaryotes, are found in almost all environments.Depending on the geographical location and season, they are represented by different morphological forms ranging from single-cell to multicellular filamentous species.
Picocyanobacteria as a group contain haploid, oligoploid, and polyploid species (Griese et al., 2011).In cyanobacteria, polyploidy has been associated with cell volume, as genome copy number is reported to be proportional to cell size (Ohbayashi et al., 2019;Watanabe, 2020).Potential benefits of polyploidy include improved chromosome repair, resistance to stress conditions, and reduced phage susceptibility (Domain et al., 2004;Watanabe, 2020;Zborowsky & Lindell, 2019).The ploidy level of cyanobacteria can be influenced by the growth phase, growth rate, and the surrounding light and nutrient conditions (Ohbayashi et al., 2019;Riaz et al., 2021;Zerulla et al., 2016).For example, marine Synechococcus strains present varying genome copy numbers under different growth conditions such as temperature and exposure to darkness, which affects the cell cycles and therefore, DNA replication (Armbrust et al., 1989;Binder & Chisholm, 1995;Liu et al., 1998;Perez-Sepulveda et al., 2018).However, the factors that determine ploidy in picocyanobacteria and how it varies among ecotypes are not well understood.
Studies of ploidy level in picocyanobacteria have mainly focused on model strains from freshwater and marine environments (e.g., Griese et al., 2011;Perez-Sepulveda et al., 2018;Zborowsky & Lindell, 2019) but information on ploidy level of brackish strains is still lacking.Freshwater picocyanobacteria can be oligoploid, containing between two and six genome copies, while several Synechocystis sp.PCC 6803 substrains are even classified as polyploid with >10 genome copies depending on the cell cycle (Griese et al., 2011;Zerulla et al., 2016).In contrast, the majority of marine Synechococcus and Prochlorococcus are haploid with a few oligoploid exceptions (Armbrust et al., 1989;Binder & Chisholm, 1995;Perez-Sepulveda et al., 2018;Zborowsky & Lindell, 2019).In this study, we investigated the genome copy number of 18 brackish picocyanobacteria from the Kalmar Algae Collection (KAC), recently isolated from the Baltic Sea Proper (7 PSU) (Aguilera et al., 2023).The Baltic Sea, one of the largest brackish water bodies on Earth, is characterized by high nutrient and DOC concentrations, and seasonal temperature variations of >15 C (Bunse et al., 2019).Baltic Sea picocyanobacteria represent a wide physiological and genetic diversity (Aguilera et al., 2023) but brackish picocyanobacteria in general remain understudied compared to their freshwater and marine counterparts.Information on genome copy numbers of brackish strains leads to a better understanding of polyploidy among picocyanobacteria and aids in the interpretation of increasing amounts of omics data.

Sample collection and culture conditions
Brackish strains used in this study represent a variety of Synechococcus ecotypes from the Baltic Sea Proper (coastal station in the Kalmar Sound 56 39 0 24.4 00 N, 16 21 0 36.6 00 E) and Linnaeus Microbial Observatory (LMO 56 55 0 512.4 00 N, 17 3 0 38.52 00 E).The brackish strains were obtained from the KAC.Intense ecophysiological characterization of the selected strains has been recently done (see details in Aguilera et al., 2023).Synechococcus strains WH8102 and WH7803 were purchased from the Roscoff Culture Collection (RCC; Roscoff, France), Cultures were grown in L1 media prepared with artificial seawater (7 PSU for brackish strains, 33 PSU for marine strains) grown at 16, 18, or 20 C and 15 μm m À2 s À1 with a light: dark cycle of 12:12 h dark cycle.All strains were harvested during the exponential phase, as previous studies have shown that the ploidy levels are often higher during this phase than in the linear or stationary phase (Griese et al., 2011).For DNA extraction, each culture was harvested and centrifuged at 8 min at 8000g.Simultaneously, samples for cell enumeration by flow cytometry were preserved in glutaraldehyde (0.25% final concentration).The cell pellet and samples for flow cytometry were stored at À80 C until further analysis.

Primer design
Commonly used rbcL primers for marine and freshwater strains (Doron et al., 2016;Griese et al., 2011) did not produce amplificons for the brackish strains.To design new primers, rbcL sequences were obtained from whole genome sequence data from strains KAC102, KAC105, KAC106, KAC108, and KAC114.The software MEGA X1 was used to visualize and align the sequences.The rbcL gene was highly conserved and degenerate primers (KAC_rbcLf: CGCGAYCG-BATGAACAAGTAY, KAC_rbcLr: CGTCGTCYTTRGT-GAAGTCSAG) were designed to target all strains.Additionally, primers from the literature (Syn_rbcL_f: TTCATCAAGAGCTGCTACGG, Syn_rbcr: GACGGC CGTACTTGTTCATC) (Doron et al., 2016) were used for the marine reference strains.All primers had an annealing temperature of 60 C and amplicon sizes between 100 and 200 bp.

DNA extraction and PCR testing
DNA was extracted from cultured KAC strains along with the two marine reference strains Synechococcus WH7803, and Synechococcus WH8102.Extraction was performed using the FastDNA™ SPIN Kit for Soil from MP Biomedicals Inc. according to the manufacturer's instructions with the addition of incubation with proteinase-K (0.02 μg μl À1 , final concentration) at 55 C for 1 h.Sample concentration was measured using an Invitrogen Qubit 2.0 fluorometer (Thermo Fisher Scientific Inc.).Sample purity was assessed using a Thermo Scientific™ NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc.).PCR reactions were performed to test for correct amplification of all primers for both reference strains and KAC strains.The PCR reaction was prepared using the Thermo Scientific Phusion High-Fidelity PCR Master Mix according to the manufacturer's instructions with a reaction volume of 25 μl.The PCR was performed on a T100™ Thermal Cycler (BIO RAD, USA) with an initial denaturation at 98 C for 30 s; 30 cycles of denaturation at 98 C for 10 s, annealing at 60 C for 1 minute, and extension at 72 C for 5 s; and a final extension step at 72 C for 2 min.A no-template control using water was included in all runs to check for contamination.

Quantitative PCR
A serial dilution was performed using brackish KAC strains and marine reference strains Synechococcus WH7803 and WH8102 to test for qPCR efficiency of primer sets KAC_rbcL and Syn strains_rbcL.A range of 1-20 ng of gDNA total input was used per reaction.The average efficiencies for each assay were, 91% for the KAC_rbcL assay and 99% for marine_Syn strains_rbcL assay.All samples used in the experiment were diluted to a final concentration of 10 ng μl À1 .qPCR reactions were prepared using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific Inc.).Each sample was run in four replicates using a 2 μl DNA template, 5 μl Master Mix, 0.3 μM of each primer, and UltraPure™ DNase/RNase-Free Distilled Water (Invitrogen™) to a final reaction volume of 10 μl.Reactions were run on a LightCycler ® 480 Instrument (Roche) with the following thermocycling settings, according to the manufacturer's instructions: 50 C for 2 min, 95 C for 2 min, 40 cycles of 95 C for 15 s followed by 60 C for 1 min, and a final step for the melt curve analysis of 95 C for 15 s, 60 C for 1 min and 95 C for 15 s.No template controls, with nuclease-free water instead of sample, were added to each primer master mix set and, on all plates, to check for contamination.Melt curve analysis showed no unspecific amplification.Genome copy numbers were calculated as described in Griese et al., 2011.Average values and standard deviations were calculated from the 4 performed replicates (Supplementary Table S1).For clarity and comparison to previous studies, we have chosen to present rounded numbers in Table 1.

Flow cytometry
All samples were run on a CYTOFlex Flow Cytometer (Beckman Coulter Inc.) equipped with a blue laser (80 mW) at 488 nm and a red laser (50 mW) at 638 nm.For each sample, 50 μl was analysed at an average flow rate of 1 μl s À1 .For the cell characterization, four optical parameters were used at a logarithmic scale: forward scatter as a proxy for cell diameter, PE (585/42 nm, blue laser dependent) as a proxy for phycoerythrin content, PC5.5 (690/50 nm, blue laser dependent) as a proxy for chlorophyll a, and APC (660/10 nm, red laser dependent) as a proxy for phycocyanin content.

RESULTS AND DISCUSSION
This study is the first report of ploidy levels of brackish picocyanobacteria.Similar to previous studies on ploidy in picocyanobacteria (e.g., Griese et al., 2011;Perez-Sepulveda et al., 2018), we used the rbcL gene to quantify the genome copy numbers using qPCR.New primers were developed due to the genomic divergence of brackish strains compared to marine and freshwater counterparts.All tested brackish strains (KAC 100-116 and KAC 125) had low variation in their genome copy number, containing one to four genome copies (Table 1).The majority (16 out of 18) were haploid or diploid, while strains KAC 115 and KAC112 were oligoploid containing four and three genome copies, respectively (Table 1).
In this study, all experiments were performed at standard growth conditions (details in Aguilera et al., 2023) and the ploidy level was assessed in the exponential growth phase.To ensure that the results were consistent with previous studies, two marine strains, Synechococcus strains WH7803 and WH8102, with previously reported ploidy levels, were used as controls (Table 1).As for other studies investigating the ploidy level by qPCR, the genome copy numbers presented in this study represent a population average with a possibility that the copy number of individual cells within a population is variable.Despite being cultivated at the same conditions, the genome copy number varied among our tested brackish strains, illustrating different strategies of ploidy level.Studies suggest that polyploidy can provide an advantage during unfavourable conditions (Lui et al. 2018;Pecoraro et al., 2011;Zerulla et al., 2016).An ecophysiological analysis of five of the brackish strains included in this study showed a large variation among the strains for example in tolerance for high salinity, but more sensitivity towards specific light intensity and temperature optimum.Other picocyanobacterial strains of the KAC collection showed a high tolerance towards low temperatures and lower salinity levels (Aguilera et al., 2023).Intensive work on marine Synechococcus isolates also indicates the existence of physiologically specialized ecotypes in closely related lineages (Mackey et al., 2017;Pittera et al., 2014;Sohm et al., 2016).When comparing the physiological diversity of brackish Synechococcus strains with their ploidy level, no correlation was found when analysing tolerance to abiotic factors as categorical (Yes/No) variables with Pearson's correlation coefficient test (ploidy/ salinity p = 0.11; ploidy/temperature p = 0.24; ploidy/ light tolerance p = 0.24), indicating that their physiological boundaries in the environment may not be linked with genome copy number.For an accurate assessment of the effect of environmental conditions on genome copy number in picocyanobacteria, further experiments under variable conditions will be needed.
Ploidy could provide ecological and evolutionary advantages, and the ploidy level can vary with environmental factors and growth phase (reviewed in Anatskaya & Vinogradov, 2022).Multiple genome copies may increase the flexibility in gene expression, which has been observed in a diploid diatom (Mock et al., 2017).Oligoploidy could therefore increase resilience to changing environmental conditions (Makarova et al., 2013;Soppa, 2013).Haploidy may be a trait consistent with living in an oligotrophic environment, where streamlining of genetic processes is advantageous when it comes to resource competition (Perez-Sepulveda et al., 2018).The brackish strains tested in this study were isolated from the central Baltic Sea, a temperate ecosystem that is highly dynamic (Aguilera et al., 2023;Lagus et al., 2007) and characterized by nitrogen limitation during summer (Alegria Zufia et al., 2021).For marine Synechococcus sp.WH7803, nutrient limitation did not affect genome copy numbers as extensively as observed in freshwater Synechocystis PCC6803 and a freshwater archeon (Haloferax volcanii).For instance, changes in phosphate (P) concentrations result in copy number variations from 4 copies in P-deplete to 35 copies in P-replete conditions in the freshwater picocyanobacteria strain PCC 6803; and 2 (P-deplete) to 20 (normal growth conditions) in Haloferax volcanii (Zerulla et al., 2014(Zerulla et al., , 2016)).With 3.7 copies in P-deplete and 4.8 copies in normal growth conditions, the marine strain Synechococcus sp.WH7803 remains more stable (Perez-Sepulveda et al., 2018).Similar patterns were observed for the genome copy number changes during different growth phases.For example, the marine Synechococcus sp.WH7803 showed more stability in ploidy level (3 to 6 copies) compared to freshwater Synechococcus elongatus PCC 7942 and PCC 6803 (glucose tolerant wild-type strain), where genome copy numbers ranged from 2 to 10, and 43 to 142, respectively (Griese et al., 2011;Perez-Sepulveda et al., 2018;Watanabe et al., 2015).
Based on 16S rRNA gene phylogeny, the brackish picocyanobacterial strains are assigned to subcluster 5.2 together with some freshwater, estuarine, and halotolerant strains, separated from the marine strains mostly assigned to subcluster 5.1 and 5.3 (Figure 1; Aguilera et al., 2023;Ahlgren & Rocap, 2012).More detailed genomic studies also showed that brackish picocyanobacterial strains contain a mixture of pathways typically found in marine and freshwater species and that they have intermediate genome sizes (Cabello-Yeves et al., 2022).We therefore hypothesized that brackish strains would have different traits compared to freshwater (oligoploid and polyploid) and marine strains (mostly haploid or oligoploid) when it comes to ploidy level.Available information on ploidy among freshwater strains is limited to Synechococcus elongatus PCC 7942 and Synechocystis PCC 6803 which do not cluster with the brackish picocyanobacteria strains (Figure 1).Based on the current information on ploidy in freshwater strains, the ploidy level was generally lower among the brackish strains and more similar to the genome copy numbers reported from marine strains (Supplementary Table S1 and Table 1).Among the tested brackish strains, variation in ploidy level was also seen between closely related strains (e.g., 100% 16S rRNA gene identity of KAC 115 [4 genome copies] and KAC 103 [1 genome copy]; Figure 1).This has previously been shown for other prokaryotes including, for example, Gammaproteobacteria (Pecoraro et al., 2011), suggesting that phylogenetic similarity may not select for a certain ploidy level and that phylogenetic analysis itself cannot reveal insights into ploidy level.
It is important to consider ploidy level when interpreting molecular data on relative abundances from environmental samples as polyploidy may lead to an overestimation of certain taxa within the community.In the Baltic Sea, multiple amplicon-based studies report significant contributions of picocyanobacteria (up to 80%) in 16S rRNA gene amplicon libraries (Andersson et al., 2010;Bertos-Fortis et al., 2016;Celepli et al., 2017;Lindh et al., 2015).Although the brackish strains generally had low genome copy numbers per cell, in this study, 22% of the brackish strains were at least diploid.However, because of the high similarity of the 16S rRNA gene among co-occurring brackish picocyanobacterial strains (Aguilera et al., 2023) and the possibility of changing genome copy numbers with different environmental conditions and growth phase (e.g., Ohbayashi et al., 2019;Riaz et al., 2021;Zerulla et al., 2016) integrating a calibration of ploidy level into amplicon sequencing data remains challenging.Further characterization both on the genomic and physiological level (e.g., growth during nutrient depletion and phage susceptibility) will be critical for understanding ploidy strategies among brackish picocyanobacteria as well as for the accurate interpretation of the growing amount of sequencing data.